Detection of Nucleic acids (such as DNA and RNA) is important in various fields and uses, including medicine and biology, such as the evaluation of the existence of microorganisms, diagnosis of infection, evaluation of gene polymorphism, and patient profiling, as well as in food examination, environment evaluation, and forensic medicine. Moreover, in recent years, many short-chain nucleic acids (such as miRNA) that perform important roles in vital activities have been discovered, and a need for a means of detecting such short-chain nucleic acids has been increasing.
PCR amplification is generally performed as a method for specifically detecting a nucleic acid with high sensitivity (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). In the PCR, a nucleic acid is amplified, for example, through a reaction consisting of three steps: dissociation (denaturation) of a double-stranded template DNA into single strands, annealing of primers to the single-stranded template DNAs, and synthesis (extension) of a complementary strand from each primer. In usual PCR, the denaturation process, the annealing process, and the extension process are carried out at different temperatures with a thermal cycler. Consequently, expensive temperature cycle control equipment is necessary to perform such a reaction, resulting in a problem that the method is prevented from being employed in field examinations, point-of-care (bedside) diagnoses, and inexpensive examinations. Moreover, since the reaction is carried out at three different temperatures, the reaction has problems that the temperature control is troublesome and that the time loss increases in proportion to the number of cycles. Additionally, the exponential amplification requires multiple primers, and a short-chain nucleic acid that cannot ensure a primer binding sequence cannot be directly detected. Accordingly, it has been proposed to carry out amplification in combination with another reaction that adds a primer sequence to the target nucleic acid as pre-processing. However, the additional process for the addition reaction makes the detection operation more complicated and take longer time, needs the reagents to be used, and also has risks of a decrease in sensitivity and of losing the quantitativity due to partial or complete loss of the sample. Furthermore, there is a risk that the quantitative ratio information between multiple samples is lost due to a variation in efficiency of the process of adding a different sequence.
Accordingly, methods for nucleic acid amplification that can be performed under isothermal conditions have been developed as alternatives to PCR. Examples of these methods include strand displacement amplification method (SDA) described in G. T. Walker, M. C. Little, J. G. Nadeau, and D. D. Shank, “Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system”, Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992) and Japanese Patent Publication No. Hei 7-114718; loop-mediated isothermal amplification (LAMP) described in International Publication No. WO 00/28082; and isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) described in International Publication No. WO 02/16639.
In the SDA described by G. T. Walker et al., detection reactions occur even in systems not containing target nucleic acids in some cases, and the evaluation results in sensitivity and specificity are therefore questionable. The SDA described in Japanese Patent Publication No. Hei 7-114718 is a system of ultimately amplifying DNA and can amplify a target nucleic acid (and its complementary strand) in a sample through displacement of a double strand mediated by a DNA polymerase and a restriction enzyme. This method needs four different primers, of which two are required to be designed so as to include the recognition site of the restriction enzyme. This method cannot be used for direct amplification of short-chain nucleic acids.
The LAMP also needs four different primers that recognize six regions and thereby can amplify a target gene. In other words, in this method, a first primer anneals to the template strand to cause an extension reaction, and then the extended strand by the first primer is separated from the template strand by a strand displacement reaction by a second primer designed upstream of the first primer. On this occasion, a stem loop structure is formed at the 5′ end portion of the extended strand due to the structure of the removed first primer extension product, and this method cannot be used for direct amplification of short-chain nucleic acids.
Additionally, in Y. Weizmann, M. K. Beissenhirtz, Z. Cheglakov, R. Nowarski, and I. Willner, “A virus spotlighted by an autonomous DNA machine”, Angew. Chem. Int. Ed., 45, 7384-7388 (2006), DNA (deoxyribozyme) having an enzyme activity is synthesized using a sequence that binds to the detection target nucleic acid as a primer in the extension reaction by a DNA polymerase having nicking ability and strand displacement ability, and then detection is carried out using the molecules generated by the peroxidase reaction of the deoxyribozyme as a signal. In also this method, short-chain nucleic acids are not detected.    Patent Literature 1: U.S. Pat. No. 4,683,195    Patent Literature 2: U.S. Pat. No. 4,683,202    Patent Literature 3: U.S. Pat. No. 4,800,159    Patent Literature 4: Japanese Patent Publication No. Hei 7-114718    Patent Literature 5: International Publication No. WO 00/28082    Patent Literature 6: International Publication No. WO 02/16639    Non-Patent Literature 1: G. T. Walker, M. C. Little, J. G. Nadeau, and D. D. Shank, “Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system”, Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992)    Non-Patent Literature 2: Y. Weizmann, M. K. Beissenhirtz, Z. Cheglakov, R. Nowarski, and I. Willner, “A virus spotlighted by an autonomous DNA machine”, Angew. Chem. Int. Ed., 45, 7384-7388 (2006)